Pyrazolo quinazoline derivative compounds inducing selective degradation of plk1

ABSTRACT

The present invention provides novel compounds that induce selective polo-like kinase 1 (PLK1) degradation. Specifically, the present invention provides a bifunctional compound in which a PLK1 binding moiety and an E3 ubiquitin ligase-binding moiety are linked by a chemical linker. The present invention provides the compound, a method for preparing the same, and the use thereof. The compounds may be effectively utilized for preventing or treating PLK1 related diseases.

TECHNICAL FIELD

The present invention relates to a selective PLK1 degradation inducing compound, a method for preparing the same, and the use thereof.

BACKGROUND ART

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in the conversion of G2/M phase during cell growth and division. PLK1 is expressed and activated in a pulse form from the S phase to the G2/M phase, and rapidly degrades as mitosis ends.

PLK1 is overexpressed in various carcinomas such as colon cancer, lung cancer, bladder cancer, and melanoma, etc., and cancer cells overexpressing PLK1 tend to show resistance to various types of anticancer drugs. As the PLK1 dependence in various carcinomas was revealed as described above, there have been attempts to develop PLK1 inhibitor compounds such as volasertib (also known as BI6727), etc.

However, the conventional PLK1 inhibitors do not sufficiently inhibit PLK1 activity at concentrations that are clinically safe. Thus, there is a problem that even if the cell cycle of cancer cells is temporarily delayed, some cancer cells eventually restart the cell cycle, which may not obtain sufficient clinical effects (see Gheghiani et al., Cell Reports, 2017, etc.). In fact, many pharmaceutical companies such as Boehringer Ingelheim, GlaxoSmithKline, etc., have attempted to develop small-molecular compound-based PLK1 inhibitors, but most of them have failed or stopped in the clinical trial stage, and thus there are no commercially available PLK1 inhibitors to date. It shows that pharmacological mechanism that follows the method of inhibiting enzyme activity by binding to the active site of PLK1 like the small molecule compound inhibitors is not sufficiently effective in the development of new drugs intended to derive anticancer effects by inhibiting PLK1 activity of cancer cells.

Recently, a proteolysis targeting chimera (PROTAC) has been proposed as a small molecule-based platform technology capable of inducing proteolysis of a target protein in the body. The PROTAC is a bifunctional compound in which a ligand molecule that binds to disease-related target protein and an E3 ubiquitin ligase binding moiety are linked by a chemical linker. Theoretically, the PROTAC compound is capable of inducing degradation of the target protein by placing the disease-related target protein near the E3 ubiquitin ligase.

In the case of the PROTAC compound using PLK1 as a target protein, Chinese Patent Laid-Open No. 106543185 A discloses some bifunctional compounds in which a volasertib derivative compound and a binding moiety for the E3 ubiquitin ligase CRBN are linked by a chemical linker. However, the related art document merely describes some limited forms of synthesis examples of PROTAC compounds, wherein in general, the target degradation activity and selectivity of PROTAC may vary significantly depending on selection of the target protein moiety, the E3 ubiquitin ligase binding moiety, and the like (see Burslem and Crews, 2017, etc.).

Further, the PROTAC compound described in the above-described document is characterized by a compound that simultaneously degrades PLK1 and BRD4, and also induces the degradation of various proteins such as PLK family proteins other than PLK1 and BRD4, etc.), which may cause side effects due to off-target effects at the time of drug development. In particular, considering that it is known that strong inhibition of BRD4 activity inevitably accompanies on-target toxicity such as blood toxicity and gastrointestinal toxicity along with pharmacological effects, the more effectively the PROTAC compound described in the above document degrades BRD4, the greater the clinical side effects are expected (see Bolden et al. Cell Reports, 2014).

Moreover, according to the document published by the inventors of the above document (see Mu et al. BBRC, 2019), it can be confirmed that the PROTAC compound, which simultaneously degrades PLK1 and BRD4, has much stronger BRD4 degradation ability than PLK1 degradation ability at the cellular level, and the cell cycle thereof almost stops in the G1 phase, etc., that is, the PROTAC compound actually acts only as a BRD4 inhibitor regardless of the way that the conventional PLK1 inhibitors exert pharmacological effects.

Therefore, there is an unsatisfied demand for a PLK1 selective degradation inducing compound, which has a selective degradation activity for PLK1 while effectively inducing the degradation of PLK1, thereby minimizing side effects.

DISCLOSURE OF INVENTION Technical Problem

An object of the present invention is to provide selective PLK1 degradation inducing compounds.

Another object of the present invention is to provide a method for preparing the compounds.

Still another object of the present invention is to provide a use of the compounds.

Solution to Problem

Selective PLK1 Degradation Inducing Compounds

The present invention provides novel compounds that induce selective polo-like kinase 1 (PLK1) degradation. Specifically, the present invention provides a bifunctional compound in which a PLK1 binding moiety and an E3 ubiquitin ligase-binding moiety are linked by a chemical linker.

In one general aspect, there is provided a compound represented by the following Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

ULM-Linker-PTM  [Formula I]

in the Formula I above,

ULM is CRBN or VHL E3 ubiquitin ligase binding moiety;

PTM is PLK1 binding moiety represented by the following Formula II:

{in the Formula II above,

R₁ is hydrogen, C₁₋₄ alkyl or —(C₁₋₃ alkyl)-OH;

R₂ is —O—C₁₋₄ alkyl or —O—CF₃;

R₃ is N or CH; and

R4 is NH, N(C₁₋₃ alkyl), CH₂ or CH(C₁₋₃ alkyl)}; and

Linker is a chemical group that links ULM and PTM.

In the Formula II,

indicates a covalent bond that links PTM into Linker.

(1) E3 Ubiquitin Ligase Binding Moiety (ULM)

In one embodiment of the present invention, ULM is a CRBN E3 ubiquitin ligase binding moiety.

In the present invention, CRBN means Cereblon E3 ubiquitin ligase. CRBN constitutes an E3 ubiquitin ligase complex together with DDB1, Cul4A and ROC1, wherein the CRBN is a substrate recognition subunit of the complex. Some compounds capable of binding to the CRBN E3 ubiquitin ligase are known in the art. For example, after it was known that thalidomide binds to the CRBN E3 ubiquitin ligase (see Ito et al. 2010), it has been reported that a number of immunomodulatory imide drugs (IMiD) including lenalidomide and pomalidomide have CRBN binding ability (see Chamberlain and Brian. 2019; Akuffo et al. 2018; and Burslem et al. 2018, etc.).

In one embodiment, the CRBN E3 ubiquitin ligase binding moiety in Formula I is represented by the following Formula A:

wherein:

X₁ is —CH₂—, —CH(C₁₋₄ alkyl)-, —CO— or —N═N—; and

X₂ is hydrogen or C₁₋₃ alkyl;

wherein

indicates a covalent bond that links ULM into Linker.

In another embodiment of the present invention, ULM is a VHL E3 ubiquitin ligase ligand binding moiety.

In the present invention, VHL means a von Hippel-Lindau tumor suppressor. VHL constitutes a VCB E3 ligation complex together with Elongin B, Elongin C, CUL2 and Rbx1, wherein VHL is a substrate recognition subunit of the complex. Some compounds capable of binding to the VHL E3 ubiquitin ligase are known in the art. For example, after it was known that peptide such as Ala-Leu-Ala-(Hy)Pro-Tyr-Ile-Pro heptapeptide (see Schneekloth et al. 2004) and Leu-Ala-(Hy)Pro-Tyr-Ile pentapeptide (see Rodriguez-Gonzalez et al. 2008), an improved low-molecular VHL E3 ubiquitin ligase binding compound has been reported (see Buckley et al. J. Am. Chem. Soc. 2012; Buckley et al. Ang. Chem. Int. Ed. 2012; Galdeano et al. 2014; Soares et al. 2017, etc.).

In one embodiment, the VHL E3 ubiquitin ligase binding moiety in Formula I is represented by the following Formula B:

wherein:

{circle around (H)} is 5-membered heteroaryl;

Y₁ is hydrogen or C₁₋₃ alkyl;

wherein

indicates a covalent bond that links ULM into Linker.

In certain example, Formula B is represented by the moiety selected from the group consisting of:

(2) Protein Target Moiety (PTM)

In the compound represented by Formula I, the PTM, a moiety that performs a target protein ligand function, is a polo-like kinase 1 (PLK1) binding moiety represented by Formula II above.

The compound represented by Formula II alone is a pyrazoloquinazoline derivative that may bind to the active site of PLK1 (see Valsasina, Barbara, et al. Molecular cancer therapeutics 11.4 (2012): 1006-1016. And WO2008/074788, etc.).

In one embodiment, Formula II is represented by the following Formula III-1 or III-2.

In the Formula III-1 and III-2, R₁, R₂, R₃ and R₄ are the same as defined in Formula II.

In certain embodiment, R₁ is CH₃.

In certain embodiment, R₂ is —OCH₃ or —OCF₃.

In certain embodiment, R₃ is N.

In certain embodiment, R₄ is NH or N(C₁₋₂ alkyl).

In one embodiment, Formula III-1 is represented by the following:

In one embodiment, Formula III-2 is represented by the following:

(3) Linker

In one embodiment of the present invention, the Linker as defined in Formula I is represented by the following Formula L:

wherein:

and ----- are each independently bond;

L_(ULM) is covalently bonded to ULM moiety through

that is linked thereto,

L_(PTM) is covalently bonded to PTM moiety through

that is linked thereto,

L_(ULM) and L_(PTM) are each independently a single bond, —CH₂—, —NH—, —O—, —CO—, —OCO—, —CONH— or —NHCO—,

L_(LNT) is selected from the group consisting of —CH₂—, —CH₂CH₂—, —CHCH—, —CC—, —CH₂CH₂O—, —OCH₂CH₂—, —CH₂CH₂S—, —SCH₂CH₂—, —COO—, —CONH—, —NHCO— and {circle around (L)} {wherein {circle around (L)} is 3-10 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl}; and

p is an integer between 1 to 10.

In one embodiment, Linker is a linker that is included in the compound selected from the group consisting of Compound 2 to 12.

In a certain embodiment of the present invention, the compound represented by Formula I is a compound that is selected from the group consisting of Compound 2 to 12.

In the present invention, a pharmaceutically acceptable salt refers to any organic or inorganic acid addition salt with a concentration that is relatively non-toxic, is harmless, and has effective action to patients, wherein side effects caused by this salt does not deteriorate beneficial efficacy of the compound represented by Formula I. For example, the pharmaceutically acceptable salt may be an inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, or the like, or an organic acid such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid or hydroiodic acid, but is not limited thereto.

Method for the Preparing the Selective PLK1 Degradation Inducing Compounds

In the present invention, the compound represented by Formula I above, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof may be prepared through reactions such as the following Reaction Schemes 1 to 3 by a synthetic method known in the field of organic chemistry or a modification technique apparent to those skilled in the art.

In the Reaction Schemes 1 to 3 above, PTM, Linker and ULM are a group defined in the above, or a suitable derivative thereof. RG¹, RG², RG^(2a) RG^(2b), RG³, RG^(3a), RG^(3b)and RG⁴ are moieties including a suitable reactive group capable of linking together with an intermediate of the PROTAC compound represented by Formula I through formation of the covalent bond in the field of organic synthesis. The formation of the covalent bond may be achieved by synthetic reactions such as amide formation, ester formation, carbamate formation, urea formation, ether formation, amine formation, and single bonds, double bond formation between various carbons, click chemistry and the like, depending on specific reaction groups, but is not limited thereto.

Variations of each step in the above Reaction Scheme may include one or multiple synthesis steps. Isolation and purification of the product may be accomplished by standard procedures known to those skilled in the art of organic chemistry.

In one embodiment, the compounds of the present invention can be prepared through Reaction Scheme 2 by one or multiple synthetic steps.

For example, when ULM is Formula A, the compound of the present invention may be prepared through the following reaction scheme.

For example, when ULM is Formula B, the compound of the present invention may be prepared through the following reaction scheme.

In the above schemes, RG¹ and RG² are L_(PTM) or any reaction precursor thereof, RG3 and RG⁴ are L_(ULM) or any reaction precursor thereof, and RG¹, RG², RG³ and RG⁴ may be appropriately selected according to the structure and linker position of the target compound.

For example, in case

RG¹ may be —COOH as shown in Examples

For example, in case

RG¹ may be hydrogen as shown in Examples 5-7.

In the above Reaction Scheme, each compound represented by PTM and ULM may be synthesized by a person skilled in the art with reference to documents known in the field of organic chemistry, descriptions of Examples of the present invention, and the like.

The present invention also provides the compounds represented by PTM-Linker-RG³ or PTM-Linker 1-RG^(2b) that are the reaction intermediates of the compounds represented by Formula I.

Use of the Selective PLK1 Degradation Inducing Compounds

An embodiment of the present invention is a composition for inducing PLK1 degradation including a compound represented by Formula I or a pharmaceutically acceptable salt thereof. The Formula I is the same as defined above.

In the experimental examples of the present invention, it was confirmed that the compounds of the present invention effectively induce the protein degradation of PLK1 (FIG. 1 ). Specifically, the compounds of the present invention (Compounds 2-12) have significantly superior protein degradability of PLK1 compared to the compounds disclosed in CN 106543185 A, which is a prior art document. Further, the compounds of the present invention have little or no degrability to BRD4, a target protein other than PLK1. Therefore, there is an advantage of minimizing side effects due to off-target degradation occurring in the compounds disclosed in the prior art documents.

The PLK1 degradation-inducing PROTAC compound of the present invention is capable of fundamentally degrading the target protein, PLK1 in view of the mechanism of action, thereby achieving an excellent PLK1 inhibitory effect as compared to the conventional PLK1 small molecule inhibitor that inhibits the simple activity of PLK1.

Accordingly, the composition including the compound represented by Formula I of the present invention or a pharmaceutically acceptable salt thereof may be effectively employed for selective degradation of PLK1.

An embodiment of the present invention is a composition for preventing or treating PLK1-related diseases including the compound represented by Formula I or the pharmaceutically acceptable salt thereof. An another embodiment of the present invention is a method for the prevention or treatment of PLK-related diseases comprising administering the composition to a subject in need thereof. The Formula I is the same as defined above.

In the present invention, the PLK1-related disease refers to any disease or condition capable of being treated, alleviated, delayed, inhibited or prevented from induction of degradation or inhibition of activity of PLK1. In an embodiment, the PLK1-related disease may be a cancer (malignant tumor), a benign tumor, a neurological disease, or other genetic or non-genetic diseases caused by excessive cell division.

The cancer includes all cancers capable of exhibiting prophylactic or therapeutic efficacy due to inhibition of PLK1 activity, and may be solid cancer or blood cancer. For example, the cancer may be one or more selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma, and the like, but is not limited thereto. The cancer includes not only primary cancer but also metastatic cancer.

The benign tumors include all benign tumors capable of exhibiting prophylactic or therapeutic efficacy due to the inhibition of PLK1 activity, such as benign tumors in pre-cancer stages, and may be solid tumors or blood tumors. For example, the tumor may be one or more selected from the group consisting of Barrett's esophagus, colon adenoma and polyp, breast fibroadenoma and cyst, monoclonal gammopathy of undetermined significance (MGUS), monoclonal lymphocytosis, and the like, but is not limited thereto.

The neurological diseases include all neurological diseases capable of exhibiting prophylactic or therapeutic efficacy due to the inhibition of PLK1 activity, and specifically, may be one or more selected from the group consisting of central nervous system disease, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, senile dementia, epilepsy, Lou Gehrig, stroke, and nerve damage and axonal degeneration-related disorders following brain or spinal cord injury, but is not limited thereto.

The pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar medicinal effects in addition to the compound represented by Formula I above, or the pharmaceutically acceptable salt thereof.

An embodiment of the present invention is a method of degrading PLK1 by administering a compound represented by Formula I or a pharmaceutically acceptable salt thereof to mammals including humans.

Another embodiment of the present invention is a method of degrading PLK1 by administering the compound represented by Formula I or the pharmaceutically acceptable salt thereof to a sample in vitro. The sample may be a cell, a cell culture, a body fluid or tissue of a mammal including a human, but is not limited thereto.

Advantageous Effects of Invention

The compound of the present invention exhibits an effect of inducing PLK1 degradation. Therefore, the pharmaceutical compound of the present invention may be effectively utilized for preventing or treating PLK1-related diseases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the western blotting results from the measurement of the protein degradability of PLK1 according to the bifunctional compound of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

The present invention provides synthetic methods for Compound 1 to 12 shown in the table below.

TABLE 1 Com- pounds Structure 1

2

3

4

5

6

7

8

9

10

11

12

The compounds of the present invention were purified according to the following method and the structure was analyzed.

Instruments

LCMS: Shimadzu LCMS-2020

NMR: BRUKER AVANCE III 400 MHz

HPLC: Shimadzu LC-20AB, Shimadzu LC-20AD, Agilent 1100 LC, Agilent 1200 LC, Agilent 1290 LC

LCSM Analysis

LCMS data were recorded with Shimadzu LCMS-2020 equipped with an electron spray ionization device. 0.0375% TFA in water (solvent A) and 0.01875% TFA in acetonitrile (solvent B) were used as mobile phases. As a column, Kinetex EVO C18 (2.1*30) mm, 5 um was used.

HPLC Analysis

In HPLC analysis, Shimadzu LC-20AB, Shimadzu LC-20AD, Agilent 1100 LC, Agilent 1200 LC or Agilent 1290 LC was used. 0.0375% TFA in water (solvent A) and 0.01875% TFA in acetonitrile (solvent B) or 0.025% NH₃.H₂O in water (solvent A) and acetonitrile (Solvent B) was used as the mobile phase. As a column, XBridge C18 (2.1*50) mm, Sum or Kinetex C18 LC column (4.6*50) mm, Sum or Eclipse plus C18 (4.6*150) mm, 3.5 um or Waters XBridge® C18 (4.6*150) mm, 3.5 μm was used.

NMR Analysis

¹H NMR spectrum was recorded with Bruker AVANCE III 400 MHz/5 mm Probe (BBO).

SFC Analysis

In SFC analysis, SHIMADZU LC-30ADsf was used, and CO₂ (solvent A) and 0.05% DEA in methanol (solvent B) or 0.05% DEA in methanol:acetonitrile (1:1) (solvent B) was used as the mobile phase. Columns were Chiralcel OD-3 50×4.6 mm, 3 um or Chiralcel OJ-3 50×4.6 mm, 3 um or Chiralpak AD-3 50×4.6 mm, 3 um or Chiralpak AS-3 50×4.6 mm, 3 um.

Example 1. Synthesis of 8-((2-methoxy-5-(4-methylpiperazin-1-yl)-3-((2-(2-(2-(phenylamino)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 1) Step 1: Synthesis of benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (26)

To a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanol (5 g, 33.51 mmol) in DCM (50 mL) was added benzyl carbonochloridate (5.72 g, 33.51 mmol, 4.76 mL) and DIPEA (8.66 g, 67.03 mmol, 11.68 mL). Then the mixture was added at 25° C. for 16 h. LCMS showed desired mass was detected. The reaction mixture was concentrated in vacuum to afford benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (9 g, crude) as a colorless oil, which was used into the next step without further purification.

Step 2: Synthesis of 2-[2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy]ethy] 4-methylbenzenesulfonate (27)

To a solution of benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (9 g, 31.77 mmol) in DCM (100 mL) was added 4-methylbenzenesulfonyl chloride (12.11 g, 63.53 mmol) and Py (5.10 g, 64.49 mmol, 5.20 mL). Then the mixture was stirred at 25° C. for 16 h. LCMS showed desired mass was detected. The reaction mixture was washed with water (100 mL×2). The organic layer was washed with brine (500 mL×2), dried over Na₂SO₄, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (80 g SepaFlash® Silica Flash Column, Eluent of 0-100% PE/EtOAc at 80 mL/min). The titled compound (8.74 g, 14.39 mmol, 45.30% yield, 72% purity) was obtained as a colorless oil. MS (M+H)⁺=438.1

Step 3: Synthesis of benzyl (2-(2-(2-(phenylamino)ethoxy)ethoxy)ethyl)carbamate (28)

To a mixture of 2-[2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (2 g, 3.29 mmol) in toluene (20 mL) was added TEA (666.11 mg, 6.58 mmol, 916.25 uL) and aniline (425.72 mg, 4.57 mmol, 417.37 uL) drop-wise at 25° C. and the resulting mixture was stirred at 120° C. for 5 h. LCMS showed major starting material and another batch aniline (306.52 mg, 3.29 mmol, 300.51 uL) was added and the resulting mixture was stirred 120° C. for 16 h. LCMS showed desired mass was detected. The reaction mixture was concentrated in vacuum. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=8/1 to 2/1) to afford the titled compound (1.1 g, 3.07 mmol, 93.24% yield) as a yellow oil.

MS (M+H)⁺=359.3

Step 4: Synthesis of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)aniline (29)

To a solution of benzyl (2-(2-(2-(phenylamino)ethoxy)ethoxy)ethyl)carbamate (900.00 mg, 2.51 mmol) in EtOAc (10 mL) was added Pd/C (50 mg, 10% purity) and the mixture was stirred at 25° C. for 16 h under H₂ (15 psi). LCMS showed the reaction was completed. The mixture was filtered and the filtrate was concentrated under vacuum to give the titled compound (530 mg, 2.36 mmol, 94.10% yield) as yellow oil.

MS (M+H)⁺=225.1

Step 5: Synthesis of ethyl (Z)-6-((dimethylamino)methylene)-1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (31)

The mixture of ethyl 1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (8 g, 36.00 mmol) and 1,1-ditert-butoxy-N,N-dimethyl-methanamine (33.92 g, 166.83 mmol, 40.00 mL) in DMF (40 mL) was stirred at 60° C. for 15 h. The mixture was concentrated. The residue was triturated by (Petroleum ether:Ethyl acetate=5:1, 100 mL), filtered. The filter cake was collected to afford the titled compound (8.5 g, 30.65 mmol, 85.15% yield) as off-white solid.

Step 6: Synthesis of ethyl 8-amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (32)

The mixture ethyl (Z)-6-((dimethylamino)methylene)-1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (8.5 g, 30.65 mmol) and guanidine; hydrochloride (3.51 g, 36.78 mmol, 2.95 mL) in EtOH (170 mL) was added NaOEt (2.71 g, 39.85 mmol) and the resulting mixture was stirred at 80° C. for 12 h. LCMS showed the reaction was completed. The mixture was filtered. The filter cake was washed with water (50 mL) and EtOH (50 mL), dried to afford the titled compound (5.6 g, 20.49 mmol, 66.85% yield) as off-white solid.

MS (M+H)⁺=274.1

¹H NMR (400 MHz, DMSO-d6) δ=8.17 (s, 1H), 6.57 (s, 1H), 4.31 (s, 3H), 4.29-4.24 (m, 2H), 2.91 (t, J=7.2 Hz, 2H), 2.73 (t, J=8.0 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H).

Step 7: Synthesis of methyl 3-bromo-2-hydroxy-5-iodobenzoate (34)

To a solution of methyl 2-hydroxy-5-iodobenzoate (55 g, 197.81 mmol) in MeOH (800 mL) was added a solution of Br 2 (47.42 g, 296.72 mmol, 15.30 mL) in MeOH (40 mL) drop-wise at 0° C. and the mixture was stirred at 25° C. for 16 h. HPLC showed that the reaction was completed. The reaction mixture was quenched with Na₂S₂O₃ (sat. aq, 500 mL), filtered. The filtered cake was collected to give the titled compound (74 g, crude) as white solid.

Step 8: Synthesis of methyl 3-bromo-5-iodo-2-methoxybenzoate (35)

To a solution of methyl 3-bromo-2-hydroxy-5-iodobenzoate (73 g, 204.52 mmol) in DMF (800 mL) was added K₂CO₃ (84.80 g, 613.55 mmol) and CH₃I (34.83 g, 245.42 mmol, 15.28 mL) drop-wise at 0° C. over a period of 30 min under N₂ and the resulting mixture was stirred at 25° C. for 12 h. TLC showed that the reaction was completed. The reaction mixture was quenched by addition of water (1600 mL) at 25° C. The aqueous phase was extracted with EtOAc (500 mL×3). The combined organic layer was washed with brine (800 mL), dried over Na₂SO₄, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (Petroleum ether:Ethyl acetate=1:10) to afford the titled compound (50 g, 134.78 mmol, 65.90% yield) as white solid.

Step 9: Synthesis of methyl 3-bromo-2-methoxy-5-(4-methylpiperazin-1-yl)benzoate (36)

The mixture of methyl 3-bromo-5-iodo-2-methoxybenzoate (19 g, 51.22 mmol), 1-methylpiperazine (5.13 g, 51.22 mmol, 5.68 mL), Pd(OAc) 2 (1.15 g, 5.12 mmol), BINAP (3.19 g, 5.12 mmol) and Cs₂CO₃ (33.38 g, 102.44 mmol) in dioxane (200 mL), and the mixture was stirred at 75° C. for 20 h under N₂ atmosphere. LCMS showed that the reaction was completed. The reaction mixture was cooled to room temperature, EtOAc (600 mL) and water (800 mL) were added and layers were separated. The aqueous phase was extracted with EtOAc (500 mL×2); the combined organic layer was washed with brine (400 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by silica gel chromatography (Ethyl acetate:Methanol=10:1) to get crude product. The crude product was diluted with water (300 mL) and adjusted pH=2 by HCl (1 M), then washed with EtOAc (250 mL×2). The aqueous layer was adjusted pH=9 by NaHCO₃ (sat. aq) and extracted with EtOAc (200 mL×3). The combined organic layer was washed with brine and dried over Na₂SO₄, filtered. The filtrate was concentrated under vacuum to afford the titled compound (13.15 g, 38.31 mmol, 74.81% yield) as yellow liquid.

Step 10: Synthesis of tert-butyl 3-bromo-2-methoxy-5-(4-methylpiperazin-1-yl)benzoate (37)

3 Batches in Parallel:

To the solution of t-BuOH (12.96 g, 174.82 mmol, 16.72 mL) in THF (50 mL) was added n-BuLi (2.5 M, 58.27 mL) at −65° C. and the resulting mixture was stirred at −65° C. for 0.5 h, a solution of methyl 3-bromo-2-methoxy-5-(4-methylpiperazin-1-yl)benzoate (5 g, 14.57 mmol) in THF (5 mL) was added and the resulting mixture was stirred at 25° C. for 3 h. LCMS showed that the reaction was completed. The mixture was poured into water (500 mL) and extracted by EtOAc (500 mL×3). The combined organic layer was washed with brine (500 mL), dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column (Petroleum ether:Ethyl acetate=1:1) to give the titled compound (10 g, 25.18 mmol, 57.60% yield, 97% purity) as yellow oil.

MS (M+H)⁺=385.1

Step 11: Synthesis of ethyl 8-((3-(tert-butoxycarbonyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (38)

To the mixture of tert-butyl 3-bromo-2-methoxy-5-(4-methylpiperazin-1-yl)benzoate (1.5 g, 3.89 mmol) and ethyl 8-amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (750 mg, 2.74 mmol) in dioxane (40 mL) was added Cs₂CO₃ (2.68 g, 8.23 mmol), [2-(2-aminophenyl)phenyl]-methylsulfonyloxy-palladium;dicyclohexyl-[3,6-dimethoxy-2-(2,4,6-triisopropylphenyl)phenyl]phosphane (248.77 mg, 274.43 umol) and the resulting mixture was stirred at 90° C. for 12 h under N₂. LCMS showed that the reaction was completed. The mixture was poured into NH₄C1 (aq. sat, 100 mL) and extracted with EtOAc (100 mL×3). The combined organic layer was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column (Ethyl acetate:Methanol=1:0-10:1) to afford the titled compound (1.5 g, 2.41 mmol, 87.99% yield, 93% purity) as yellow solid.

MS (M+H)⁺=578.4

Step 12: Synthesis of 8-((3-(tert-butoxycarbonyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid (39)

To a solution of ethyl 8-((3-(tert-butoxycarbonyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (2 g, 3.46 mmol) in THF (20 mL) and MeOH (20 mL) was added a solution of NaOH (166.17 mg, 4.15 mmol) in H₂O (40 mL) and the resulting mixture was stirred at 25° C. for 12 h. LCMS showed that the reaction was completed. The mixture was adjusted pH around 7 by HCl (1 M) and concentrated to afford the titled compound (1.9 g, 3.46 mmol, 99.85% yield) as yellow solid.

MS (M+H)⁺=550.2

Step 13: Synthesis of tert-butyl 3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-2-methoxy-5-(4-methylpiperazin-1-yl)benzoate (40)

To the mixture of 8-((3-(tert-butoxycarbonyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid (1.9 g, 3.46 mmol) in DMF (40 mL) was added EDCI (1.72 g, 8.99 mmol) and HOBt (1.17 g, 8.64 mmol) and the resulting mixture was stirred at 25° C. for 10 min, DIPEA (1.56 g, 12.10 mmol, 2.11 mL) and NH₄C1 (1.11 g, 20.74 mmol) were added and the resulting mixture was stirred at 25° C. for 12 h. LCMS showed that the reaction was completed. The mixture was filtered, the filtrate was poured into water (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layer was washed by brine (50 mL), dried over Na₂SO₄, filtered. The filtrate was concentrated to give the titled compound (1.5 g, 2.73 mmol, 79.09% yield) as yellow solid. MS (M+H)⁺=549.2

Step 14: Synthesis of 3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-2-methoxy-5-(4-methylpiperazin-1-yl)benzoic acid (41)

To the solution of tert-butyl 3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-2-methoxy-5-(4-methylpiperazin-1-yl)benzoate (1.6 g, 2.92 mmol) in dioxane (15 mL) was added HCl/dioxane (4 M, 15 mL) and the resulting mixture was stirred at 25° C. for 12 h. LCMS showed that the reaction was completed. The suspensions was filtered and filtered cake was collected to afford the titled compound (1.7 g, crude, HCl salt) as yellow solid.

MS (M+H)⁺=493.2

Step 15: Synthesis of 8-((2-methoxy-5-(4-methylpiperazin-1-yl)-3-((2-(2-(2-(phenylamino)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 1)

To the solution of 3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-2-methoxy-5-(4-methylpiperazin-1-yl)benzoic acid (190 mg, 359.17 umol, HCl salt), HATU (273.14 mg, 718.35 umol) in DMF (5 mL) was added DIPEA (139.26 mg, 1.08 mmol, 187.68 uL) and the resulting mixture was stirred at 25° C. for 30 min, N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)aniline (88.62 mg, 395.09 umol) was added and the resulting mixture was stirred at 25° C. for 0.5 h. LCMS showed that the reaction was completed. The mixture was poured into water (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layer was washed with brine (50 mL), dried over Na₂SO₄, filtered and concentrated. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*40 mm*15 um; mobile phase: [water (0.225% FA)-ACN]; B %: 11%-41%, 10 min) to 26.9 mg of desired product and 110 mg of crude product, the crude product was prep-TLC (Ethyl acetate:Methanol=10:1) to get desired product, the product was dissolved in H₂O/CH₃CN=10/1 (2 mL), the solution was adjusted PH=5 by HCOOH, then lyophilized to afford another 25 mg of desired product. Totally 8-((2-methoxy-5-(4-methylpiperazin-1-yl)-3-((2-(2-(2-(phenylamino)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (51.9 mg, 64.11 umol, 17.85% yield, 92% purity, FA salt) was obtained as gray solid.

MS (M+H)⁺=699.2

Example 2. Synthesis of 8-((3-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy) ethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 2)

To the solution of 3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-2-methoxy-5-(4-methylpiperazin-1-yl)benzoic acid (250 mg, 472.60 umol, HCl salt) in DMF (5 mL) was added HATU (359.39 mg, 945.20 umol) and DIPEA (183.24 mg, 1.42 mmol, 246.95 uL) and the mixture was stirred at 25° C. for 10 min, 4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (270.87 mg, 614.38 umol, HCl salt) was added and the resulting mixture was stirred at 25° C. for 1 h. LCMS showed that the reaction was completed. The mixture was poured into water (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layer was washed with brine (50 mL), dried over Na₂SO₄, filtered and concentrated. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150*50 mm*10 um; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B %: 17%-47%, 11.5 min) followed by prep-HPLC (column: Phenomenex Gemini-NX C18 75*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 25%-35%, 7 min) to afford 8-((3-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (39.7 mg, 38.38 umol, 8.12% yield, 96% purity, TFA salt) as yellow solid.

MS (M+H)⁺=879.4

¹H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 9.68 (br s, 1H), 8.55 (s, 1H), 8.38 (s, 1H), 8.30 (t, J=5.4 Hz, 1H), 7.66 (d, J=3.0 Hz, 1H), 7.58-7.48 (m, 1H), 7.38 (s, 1H), 7.30 (s, 1H), 7.10 (d, J=8.6 Hz, 1H), 7.03-6.92 (m, 2H), 6.59 (t, J=5.8 Hz, 1H), 5.03 (dd, J=5.4, 12.8 Hz, 1H), 4.20 (s, 3H), 3.74 (d, J=12.6 Hz, 2H), 3.68 (s, 3H), 3.64-3.53 (m, 10H), 3.51-3.45 (m, 5H), 3.15 (d, J=10.4 Hz, 2H), 3.01-2.94 (m, 2H), 2.92-2.76 (m, 8H), 2.59-2.50 (m, 1H), 2.06-1.94 (m, 1H).

Example 3. Synthesis of 8-((3-((2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl) amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 3)

In a similar manner to the above-described examples, the titled compound (274 mg, 245.73 umol, 32.50% yield, 93% purity, TFA salt) as yellow solid was obtained.

MS (M+H)⁺=923.4

¹H NMR (400 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.24 (s, 1H), 8.08 (s, 1H), 7.56-7.44 (m, 2H), 7.31 (s, 1H), 7.08 (d, J=6.8 Hz, 1H), 6.88 (d, J=8.6 Hz, 2H), 6.00 (s, 1H), 4.91 (dd, J=11.8, 5.7 Hz, 1H), 3.99 (s, 3H), 3.78 (s, 3H), 3.72-3.65 (m, 8H), 3.65-3.58 (m, 11H), 3.47-3.33 (m, 4H), 3.19 (t, J=7.5 Hz, 2H), 3.06-2.95 (m, 2H), 2.95-2.83 (m, 6H), 2.79-2.70 (m, 2H), 2.16-2.07 (m, 1H).

Example 4. Synthesis of 8-((3-((14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 4)

In a similar manner to the above-described examples, the titled compound (104 mg, 96.79 umol, 25.60% yield, 90% purity) as yellow solid was obtained.

MS (M+H)⁺=967.0

¹H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.71 (s, 1H), 8.62 (s, 1H), 8.39 (s, 1H), 8.32 (t, J=5.5 Hz, 1H), 7.70-7.63 (m, 1H), 7.56 (dd, J=8.6, 7.0 Hz, 1H), 7.39 (s, 1H), 7.30 (s, 1H), 7.11 (d, J=8.6 Hz, 1H), 7.08-6.97 (m, 2H), 6.58 (s, 1H), 5.05 (dd, J=12.9, 5.4 Hz, 1H), 4.21 (s, 3H), 3.74 (d, J=13.0 Hz, 2H), 3.68 (s, 3H), 3.59 (t, J=5.4 Hz, 2H), 3.56-3.49 (m, 12H), 3.49-3.47 (m, 4H), 3.46-3.41 (m, 4H), 3.23-3.10 (m, 2H), 3.01-2.90 (m, 4H), 2.90-2.85 (m, 4H), 2.84-2.78 (m, 2H), 2.64-2.51 (m, 2H), 2.07-1.96 (m, 1H).

Example 5. Synthesis of 8-((5-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 5)

Step 1: Synthesis of (Z)-ethyl 6-((dimethylamino)methylene)-1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (3)

A mixture of ethyl 1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (2 g, 9.00 mmol) in 1,1-di-tert-butoxy-N,N-dimethylmethanamine (3.39 g, 16.68 mmol, 4.00 mL) was stirred at 60° C. for 16 h. LCMS showed starting material was consumed completely and 89% peak with desired mass was detected. The reaction mixture was concentrated to afford (Z)-ethyl 6-((dimethylamino)methylene)-1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (2.5 g, crude) as a green solid.

Step 2: Synthesis of ethyl 8-amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (4)

To a solution of (Z)-ethyl 6-((dimethylamino)methylene)-1-methyl-7-oxo-4,5,6,7-tetrahydro-1H-indazole-3-carboxylate (2.1 g, 7.57 mmol) and Guanidine (2.17 g, 22.72 mmol, HCl) in EtOH (21 mL) was added NaOEt (1.55 g, 22.72 mmol) and the mixture was stirred at 80° C. for 14 h. LCMS showed the starting material remained and the mixture was stirred at 85° C. for another 14 h. LCMS showed 18% of the starting material remained and 61% of the desired mass was detected. The reaction was concentrated under reduced pressure. The crude was diluted with MeOH (10 mL), EtOAc (10 mL) and MTBE (10 mL), then filtered. The filter cake was washed with EtOAc (20 mL), the filter cake was collected and dried in vacuo to afford ethyl 8-amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (2 g, 6.81 mmol, 89.88% yield, 93% purity) as a yellow solid. MS (M+H)⁺=274.1.

Step 3: Synthesis of tert-butyl 4-(3-bromo-4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate (5)

To a solution of 2-bromo-4-iodo-1-(trifluoromethoxy)benzene (5 g, 13.63 mmol) and tert-butyl piperazine-1-carboxylate (2.75 g, 12.35 mmol) in dioxane (200 mL) were added Cs₂CO₃ (13.32 g, 40.88 mmol), BINAP (254.57 mg, 408.83 μmol) and Pd(OAc)₂ (61.19 mg, 272.55 μmol) and the mixture was stirred at 60° C. for 14 h. TLC (Petroleum ether:Ethyl acetate=5:1) showed new spot was formed. The mixture was diluted with EtOAc (50 mL) and filtered. The filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (40 g SepaFlash® Silica Flash Column, Eluent of 0-20% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to afford tert-butyl 4-(3-bromo-4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate (0.6 g, 1.31 mmol, 9.63% yield, 93% purity) as a yellow solid.

Step 4: Synthesis of ethyl 8-((5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethoxy) phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (6)

To a solution of ethyl 8-amino-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (130.00 mg, 475.69 umol) and tert-butyl 4-(3-bromo-4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate (200 mg, 470.32 mol) in dioxane (4 mL) were added Cs₂CO₃ (459.72 mg, 1.41 mmol) and BrettPhosPdG₃ (42.63 mg, 47.03 μmol) and the mixture was stirred at 100° C. for 14 h. LCMS showed the desired mass was detected. The mixture was diluted with EtOAc (10 mL) and filtered. The filter cake was washed with EtOAc (20 mL), the filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (5 g SepaFlash® Silica Flash Column, Eluent of 10-40% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to afford ethyl 8-((5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-m ethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (140 mg, 219.88 mol, 46.75% yield, 97% purity) as a yellow solid. MS (M+H)⁺=618.3.

Step 5: Synthesis of 8-((5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethoxy) phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid

To a solution of ethyl 8-((5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethoxy) phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylate (140 mg, 226.68 μmol) in THF (0.7 mL) and MeOH (0.7 mL) was added NaOH (3 M, 150 μL) and the mixture was stirred at 30° C. for 14 h. LCMS showed the desired mass was detected. The reaction was concentrated under reduced pressure to afford 8-((5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-m ethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid (150 mg, crude) as a yellow solid. MS (M+H)⁺=590.3.

Step 6: Synthesis of tert-butyl 4-(3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate (8)

To a solution of 8-((5-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-(trifluoromethoxy) phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid (150 mg, 245.28 μmol) and NH₄C1 (65.60 mg, 1.23 mmol) in DMF (3 mL) were added EDCI (70.53 mg, 367.92 μmol), HOBt (49.71 mg, 367.92 μmol) and DIPEA (158.50 mg, 1.23 mmol, 213.62 μL) and the mixture was stirred at 20° C. for 14 h. LCMS showed the starting material remained and the desired mass was detected. The mixture was stirred at 30° C. for another 14 h. LCMS showed the starting material was consumed and the desired mass was detected. The mixture was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layer was washed with brine (10 mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure to afford tert-butyl 4-(3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate (130 mg, 220.87 μmol, 90.05% yield) as a yellow solid. MS (M+H)⁺=589.2.

Step 7: Synthesis of 1-methyl-8-((5-(piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (9)

To a solution of tert-butyl 4-(3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo [4,3-h]quinazolin-8-yl)amino)-4-(trifluoromethoxy)phenyl)piperazine-1-carboxylate (130 mg, 220.87 μmol) in dioxane (2 mL) was added HCl/dioxane (4 M, 2 mL) and the mixture was stirred at 20° C. for 1 h. LCMS showed the desired mass was detected after work-up. The mixture was concentrated under reduced pressure to afford 1-methyl-8-((5-(piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (130 mg, crude, 2HCl) as a yellow solid. MS (M+H)⁺=489.1.

Step 8: Synthesis of 8-((5-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 5)

To a solution of 2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (132 mg, 235.89 umol) and 1-methyl-8-((5-(piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (110 mg, 195.94 umol, 2HCl) in dioxane (2 mL) were added K₂CO₃ (135.41 mg, 979.72 umol) and NaI (14.68 mg, 97.97 umol) and the mixture was stirred at 80° C. for 28 h. LCMS showed the desired mass was detected. The mixture was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layer was washed with H₂O (10 mL), dried over Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Unisil 3-100 C18 Ultra 150*50 mm*3 um; mobile phase: [water (0.225% FA)-ACN]; B %: 20%-50%, 10 min) and the eluent was lyophilized to afford 8-((5-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (26 mg, 27.36 umol, 13.96% yield, 97% purity, FA salt) as a yellow solid. MS (M+H)⁺=876.5.

¹H NMR (400 MHz, CDCl₃) δ=9.19-9.11 (m, 1H), 8.32 (s, 1H), 8.03-8.00 (m, 1H), 7.56-7.44 (m, 1H), 7.23 (s, 1H), 7.17-7.13 (m, 1H), 7.11 (d, J=7.0 Hz, 1H), 6.91 (d, J=8.6 Hz, 1H), 6.85-6.79 (m, 1H), 6.58-6.52 (m, 2H), 5.42-5.37 (m, 1H), 4.94-4.88 (m, 1H), 4.33 (s, 3H), 3.78-3.72 (m, 4H), 3.70-3.66 (m, 4H), 3.50-3.44 (m, 2H), 3.32-3.25 (m, 4H), 3.16 (t, J=7.6 Hz, 2H), 2.92-2.67 (m, 11H), 2.16-2.09 (m, 1H).

Example 6. Synthesis of 8-((5-(4-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-m ethyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 6)

According to the reaction scheme, the titled compound (21.7 mg, 21.23 μmol, 11.92% yield, 90% purity) as a yellow solid was obtained.

MS (M+H)⁺=920.7.

¹H NMR (400 MHz, CD₃OD) δ=8.29 (s, 1H), 7.62 (d, J=2.9 Hz, 1H), 7.51-7.46 (m, 1H), 7.20-7.16 (m, 1H), 7.01 (t, J=8.0 Hz, 2H), 6.74-6.69 (m, 1H), 5.05-4.99 (m, 1H), 4.24 (s, 3H), 3.71-3.68 (m, 2H), 3.68-3.64 (m, 8H), 3.62-3.59 (m, 2H), 3.47-3.43 (m, 2H), 3.23-3.17 (m, 4H), 3.09-3.03 (m, 2H), 2.89-2.84 (m, 2H), 2.83-2.77 (m, 1H), 2.75-2.62 (m, 8H), 2.11-2.02 (in, 1H)

Example 7. Synthesis of 8-((5-(4-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)piperazin-1-yl)-2-(trifluoromethoxy)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 7)

According to the reaction scheme, the titled compound (8 mg, 8.05 μmol, 6.46% yield, 97% purity) as a white solid was obtained.

MS (M+H)⁺=964.4.

¹H NMR (400 MHz, CD₃OD) δ=8.29 (s, 1H), 7.63 (d, J=3.0 Hz, 1H), 7.52-7.47 (m, 1H), 7.21-7.17 (m, 1H), 7.02 (t, J=7.5 Hz, 2H), 6.75-6.71 (m, 1H), 5.05-5.00 (m, 1H), 4.25 (s, 3H), 3.69-3.58 (m, 16H), 3.46-3.42 (m, 2H), 3.23-3.20 (m, 4H), 3.09-3.03 (m, 2H), 2.88-2.84 (m, 2H), 2.84-2.78 (m, 1H), 2.75-2.64 (m, 8H), 2.12-2.03 (m, 1H).

Example 8. Synthesis of 8-((3-((2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 8) Step 1: Synthesis of tert-butyl (2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)carbamate (43a)

To a solution of 2-[2-(tert-butoxycarbonylamino)ethoxy]acetic acid (516.37 mg, 2.36 mmol) in DMF (10 mL) was added DIPEA (553.48 mg, 4.28 mmol, 745.92 uL) and HATU (895.57 mg, 2.36 mmol). The mixture was stirred at 15° C. for 20 min and a solution of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl)pyrrolidine-2-carboxamide (1 g, 2.14 mmol, HCl salt) in DMF (10 mL) with DIPEA (553.48 mg, 4.28 mmol, 745.92 uL) was added. The mixture was stirred at 15° C. for 1 hr. LCMS showed starting material was consumed completely and 88% desired mass was detected.

TLC (SiO₂, Dichloromethane:Methanol=10:1) indicated starting material was consumed completely and one new spot was detected. The reaction mixture was diluted with H₂O (60 mL) and extracted with EtOAc (60 mL×3). The organic layer was washed with brine (60 mL×3). dried over Na₂SO₄, filtrated and concentrated. The residue was purified by column chromatography (SiO₂, Dichloromethane/Methanol=0/1 to 10/1). Compound tert-butyl (2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)carbamate (816 mg, 1.19 mmol, 55.49% yield, 92% purity) was obtained as a yellow oil.

MS (M+H)⁺=632.2

Step 2: Synthesis of tert-butyl (2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate (43b)

In a similar manner to step 1, the titled compound (850 mg, 1.14 mmol, 62.88% yield, 91% purity) as a yellow solid was obtained.

MS (M+H)⁺=676.3

Step 3: Synthesis of tert-butyl ((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)carbamate (43c)

In a similar manner to step 1, the titled compound (1.3 g, 1.48 mmol, 86.83% yield, 82% purity) as a yellow solid was obtained.

MS (M+H)⁺=720.3

Step 4: Synthesis of tert-butyl ((S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)carbamate (43d)

In a similar manner to step 1, the titled compound (0.45 g, 0.59 mmol, 36.14% yield) as a brown oil was obtained.

MS (M+H)⁺=764.2

Step 5: Synthesis of (9H-fluoren-9-yl)methyl ((S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosyl)carbamate (43e)

To a solution of 2-[2-[2-[2-[2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (1 g, 1.93 mmol) in DMF (5 mL) was added DIPEA (553.48 mg, 4.28 mmol, 745.92 uL) and HATU (895.57 mg, 2.36 mmol). The mixture was stirred at 15° C. for 20 min and a solution of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl) benzyl)pyrrolidine-2-carboxamide (1 g, 2.14 mmol, HCl salt) in DMF (5 mL) with DIPEA (553.48 mg, 4.28 mmol, 745.92 uL) was added. The mixture was stirred at 15° C. for 12 h. LCMS showed starting material was consumed completely and desired mass was detected. TLC (SiO₂, Dichloromethane:Methanol=20:1) indicated starting material was consumed completely and three new spots were detected. The reaction mixture was diluted with H₂O (20 mL) and extracted with EtOAc (20 mL×3). The organic layer was washed with brine (20 mL×3), dried over Na₂SO₄, filtrated and concentrated. The residue was purified by column chromatography (SiO₂, Dichloromethane:Methanol=1/0 to 10/1) to afford the titled compound (552 mg, 593.48 umol, 27.72% yield) as a red oil.

MS (M+H)⁺=930.4

Step 6: Synthesis of (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (44a)

To a mixture of tert-butyl (2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)carbamate (816 mg, 1.29 mmol) in dioxane (4 mL) was added HCl/dioxane (4 M, 16 mL) in one portion at 25° C. The mixture was stirred at 25° C. for 12 h. LCMS showed starting material was consumed completely and 95% desired mass was detected. The reaction mixture was concentrated in vacuum. Compound (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (734 mg, crude, HCl salt) was obtained as a yellow solid, which was used in the next step.

MS (M+H)⁺=532.3

Step 7: Synthesis of (2S,4R)-1-((S)-2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (44b)

In a similar manner to step 6, the tilted compound (330 mg, 539.06 umol, 42.86% yield, 100% purity, HCl salt) as a yellow oil was obtained.

MS (M+H)⁺=576.3

Step 8: Synthesis of (2S,4R)-1-((S)-14-amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydr oxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (44c)

In a similar manner to step 6, the tilted compound (660 mg, 975.57 umol, 54.02% yield, 97% purity, HCl salt) as a yellow oil was obtained.

MS (M+H)⁺=620.3

Step 9: Synthesis of (2S,4R)-1-((S)-17-amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-3-azaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (44d)

In a similar manner to step 6, the tilted compound (0.4 g, 0.57 mmol, 96.97% yield, HCl) as a yellow oil was obtained. The crude product was used directly in the next step.

MS (M+H)⁺=664.2

Step 10: Synthesis of (2S,4R)-1-((S)-20-amino-2-(tert-butyl)-4-oxo-6,9,12,15,18-pentaoxa-3-azaicosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (44e)

To a solution of (9H-fluoren-9-yl)methyl ((S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosyl)carbamate (550 mg, 591.33 umol) in THF (5 mL) was added a solution of DBU (180.05 mg, 1.18 mmol, 178.26 uL) in THF (1 mL) drop-wise at 0° C. The mixture was stirred at 15° C. for 1 h. LCMS showed starting material was consumed completely and desired mass was detected. The reaction mixture was concentrated in vacuum. Added HCl (1 M) to this residue and washed with EtOAc (10 mL×4), the EtOAc layer was discarded. The aqueous phase was added NaHCO₃ (sat. aq) to adjust the pH to 8 and purified by prep-HPLC (column: Waters Xbridge C18 150*50 mm*10 um; mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN]; B %: 15%-45%, 11.5 min). The titled compound (438 mg, 581.63 umol, 98.36% yield, 94% purity) was obtained as a yellow oil.

MS (M+H)⁺=708.4

Step 11: Synthesis of 8-((3-((2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-ylphenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 8)

To a solution of 3-((3-carbamoyl-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-yl)amino)-2-methoxy-5-(4-methylpiperazin-1-yl)benzoic acid (204.84 mg, 387.24 umol, HCl salt) in DCM (4 mL) was added DIPEA (91.00 mg, 704.07 umol, 122.64 uL) and HATU (147.24 mg, 387.24 umol). The mixture was stirred at 15° C. for 20 min and a solution of (2S,4R)-1-((S)-2-(2-(2-aminoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (200 mg, 352.03 umol, HCl salt) in DCM (4 mL) with DIPEA (91.00 mg, 704.07 umol, 122.64 uL) was added. The mixture was stirred at 15° C. for 1 h. LCMS showed starting material was consumed completely and 61% desired mass was detected. The reaction mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*40 mm*15 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 12%-42%, 11 min). Compound 8-((3-((2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (118.8 mg, 103.93 umol, 29.52% yield, 98% purity, TFA salt) was obtained as a white solid, which is checked by SFC (retention time: 1.544, SFC analysis method: “Column: Chiralcel OJ-3 50; A4.6 mm I.D., 3 um Mobile phase: Phase A for CO₂, and Phase B for MeOH (0.05% DEA); Gradient elution: MeOH (0.05% DEA) in CO₂ from 5% to 40%; Flow rate: 3 mL/min; Detector: PDA; Column Temp: 35 C; Back Pressure: 100 Bar”).

MS (M+H)⁺=1006.7

¹H NMR (400 MHz, DMSO-d₆) δ=9.75 (br s, 1H), 8.96 (s, 1H), 8.61-8.51 (m, 2H), 8.38 (s, 2H), 7.74-7.68 (m, 1H), 7.49 (d, J=9.4 Hz, 1H), 7.42-7.38 (m, 4H), 7.30 (br s, 1H), 7.00-6.95 (m, 1H), 4.56 (d, J=9.5 Hz, 1H), 4.46-4.39 (m, 2H), 4.38-4.33 (m, 2H), 4.25 (d, J=5.4 Hz, 1H), 4.21 (s, 3H), 4.02 (d, J=4.2 Hz, 2H), 3.76-3.72 (m, 2H), 3.70 (s, 3H), 3.68-3.58 (m, 5H), 3.54 (br s, 2H), 3.19-3.12 (m, 2H), 3.01-2.92 (m, 4H), 2.88 (br s, 3H), 2.84-2.79 (m, 2H), 2.44 d, J=3.0 Hz, 1H), 2.41 (s, 3H), 2.09-2.02 (m, 1H), 1.90-1.80 (m, 1H), 0.93 (s, 9H).

Example 9. Synthesis of 8-((3-((2-(2-(2-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4, 5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 9)

In a similar manner to Example 8, the titled compound (119.1 mg, 107.73 umol, 32.98% yield, 95% purity) was obtained as a yellow solid.

SFC (Retention time=2.065 min, SFC analysis method: Column: Chiralcel OD-3 50×4.6 mm I.D., 3 um Mobile phase: Phase A for CO₂, and Phase B for MeOH (0.05% DEA); Gradient elution: 40% MeOH (0.05% DEA) in CO₂ Flow rate: 3 mL/min; Detector: PDA column Temp: 35° C.; Back Pressure: 100 Bar).

MS (M+H)⁺=1050.9

¹H NMR (400 MHz, Chloroform-d) δ 8.66 (s, 1H), 8.45 (t, J=5.6 Hz, 1H), 8.27 (s, 1H), 8.02-7.97 (m, 1H), 7.74 (s, 1H), 7.58 (d, J=9.4 Hz, 1H), 7.35-7.27 (m, 4H), 7.04-6.99 (m, 1H), 6.92 (d, J=6.3 Hz, 1H), 6.76 (s, 1H), 5.42 (s, 1H), 4.66 (d, J=9.4 Hz, 1H), 4.57-4.48 (m, 2H), 4.31 (s, 4H), 4.20 (dd, J=15.1, 5.1 Hz, 1H), 4.00 (s, 3H), 3.84-3.58 (m, 12H), 3.47-3.29 (m, 5H), 3.22-3.07 (m, 2H), 2.99-2.90 (m, 2H), 2.86 (t, J=7.6 Hz, 3H), 2.61 (s, 3H), 2.50 (s, 3H), 2.38-2.30 (m, 1H), 2.08-2.00 (m, 1H), 0.94 (s, 9H).

Example 10. Synthesis of 8-((3-(((S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 10)

In a similar manner to Example 8, the titled compound (160 mg, 138.90 umol, 30.38% yield, 95% purity) was obtained as a yellow solid.

SFC (Retention time=1.770 min, SFC analysis method: Column: Chiralcel OJ-3 50×4.6 mm I.D., 3 um Mobile phase: Phase A for CO₂, and Phase B for MeOH (0.05% DEA); Gradient elution: MeOH (0.05% DEA) in CO₂ from 5% to 40% Flow rate: 3 mL/min; Detector: PDA. Column Temp: 35 C; Back Pressure: 100 Bar).

MS (M+H)⁺=1094.2

¹H NMR (400 MHz, Chloroform-d) δ 8.66 (s, 1H), 8.28 (s, 1H), 8.12-8.03 (m, 2H), 7.62 (t, J=6.1 Hz, 1H), 7.54 (s, 1H), 7.35 (s, 4H), 7.19-7.16 (m, 1H), 6.77 (s, 1H), 5.48 (s, 1H), 4.65 (t, J=8.0 Hz, 1H), 4.61-4.50 (m, 3H), 4.38-4.32 (m, 1H), 4.30 (s, 3H), 4.03-3.92 (m, 2H), 3.90-3.83 (m, 1H), 3.77 (s, 3H), 3.70-3.63 (m, 8H), 3.63-3.56 (m, 7H), 3.40-3.34 (m, 4H), 3.14 (t, J=7.6 Hz, 2H), 2.94-2.81 (m, 6H), 2.56 (s, 3H), 2.50 (s, 3H), 2.48-2.40 (m, 1H), 2.16-2.09 (m, 1H), 0.95 (s, 9H).

Example 11. Synthesis of 8-((3-(((S)-16-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 11)

In a similar manner to Example 8, the titled compound (83 mg, 72.18 umol, 12.21% yield, 99% purity) was obtained as a white solid, which is checked SFC (retention time=2.160, analysis method: “Column: Chiralpak AD-3 50×4.6 mm I.D., 3 um; Mobile phase: Phase A for CO₂, and Phase B for MeOH+ACN (0.05% DEA); Gradient elution: 50% MeOH+CAN (0.05% DEA) in CO₂; Flow rate: 3 mL/min; Detector: PDA; Column Temp: 35 C; Back Pressure: 100 Bar”).

M (M+H)⁺=1138.5

¹H NMR (400 MHz, CDCl₃) δ=8.68 (s, 1H), 8.32 (s, 1H), 8.11-8.05 (m, 2H), 7.51 (s, 1H), 7.43 (t, J=5.6 Hz, 1H), 7.39-7.33 (m, 4H), 7.26-7.22 (m, 2H), 6.78 (br s, 1H), 5.42 (br s, 1H), 4.73 (t, J=7.9 Hz, 1H), 4.61-4.49 (m, 3H), 4.37 (d, J=5.3 Hz, 1H), 4.33 (s, 3H), 4.09 (d, J=11.2 Hz, 1H), 4.04-3.91 (m, 2H), 3.80-3.79 (m, 3H), 3.68 (d, J=2.0 Hz, 5H), 3.65-3.59 (m, 13H), 3.30-3.22 (m, 4H), 3.19-3.14 (m, 2H), 2.88 (t, J=7.8 Hz, 2H), 2.63 (br s, 4H), 2.56 (dd, J=4.8, 8.2 Hz, 1H), 2.52 (s, 3H), 2.40 (s, 3H), 2.14 (dd, J=8.2, 13.4 Hz, 1H), 0.95 (s, 9H).

Example 12. Synthesis of 8-((3-(((S)-19-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-azahenicosyl)carbamoyl)-2-methoxy-5-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide (Compound 12)

In a similar manner to Example 8, the titled compound (147.2 mg, 115.78 umol, 27.84% yield, 93% purity) was obtained as a white solid, which is checked by SFC (retention time: 2.230, SFC analysis method: “Column: Chiralpak AS-3 50×4.6 mm I.D., 3 um; Mobile phase: Phase A for CO₂, and Phase B for MeOH (0.05% DEA); Gradient elution: MeOH (0.05% DEA) in CO₂ from 5% to 40%; Flow rate: 3 mL/min; Detector: PDA; Column Temp: 35 C; Back Pressure: 100 Bar”).

MS (M+H)⁺=1182.8

¹H NMR (400 MHz, CDCl₃) δ=8.67 (s, 1H), 8.32 (s, 1H), 8.11-8.05 (m, 2H), 7.49 (s, 1H), 7.42 (br s, 1H), 7.38-7.33 (m, 4H), 7.27-7.22 (m, 2H), 6.79 (br s, 1H), 5.50 (br s, 1H), 4.73 (t, J=8.0 Hz, 1H), 4.60-4.50 (m, 3H), 4.38-4.34 (m, 1H), 4.33 (s, 3H), 4.10-4.04 (m, 1H), 4.01-3.90 (m, 2H), 3.79 (s, 3H), 3.68 (s, 4H), 3.66-3.59 (m, 18H), 3.27-3.22 (m, 4H), 3.18-3.13 (m, 2H), 2.90-2.85 (m, 2H), 2.64-2.55 (m, 4H), 2.55-2.48 (m, 4H), 2.37 (s, 3H), 2.14 (dd, J=8.0, 13.5 Hz, 1H), 0.95 (s, 9H).

Comparative Example 1. Exemplary compound described in CN 106543185 A (Comparative Compound 1)

Comparative Example 2. Exemplary compound described in CN 106543185 A (Comparative Compound 2)

Experimental Examples

1. Culture of HeLa Cell Line

The HeLa cell line was purchased from Korea Cell Line Bank (KCLB), Seoul, Korea. The passage in cell culture was maintained at P115 to P125.

For cell counting, cell counter (Thermo Fisher Scientific Inc., Catalog #AMQAX1000) and 0.4% trypan blue solution were used.

For cell culture, DMEM (Gibco, Cat. No. 1195-65; Lot. No. 2085318), FBS (Gibco, Cat. No. 16000-044; Lot. No. 2097593), Penicillin/Streptomycin (PS) (Gibco, Cat. No. 15140-122; Lot. No. 2058855), 100 mm 2 cell culture dish (SPL, Cat. No. 20100), 150 mm 2 cell culture dish (SPL, Cat. No. 20150), 12-well culture plate (SPL, Cat. No. 30012), PBS pH 7.4 (Gibco, Cat. No. 10010-023; Lot. No. 2085080), TrypLE™ Express (Gibco, Cat. No. 12605-010; Lot No. 2070638), Counting Chamber (Hematocytometer) (Hirschmann, Cat. No. 8100204), and 0.4% Trypan Blue Solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20190723) were used.

2. Treatment of Compounds of the Present Invention

2×10⁵ cells were seeded for each well of a 12-well plate (SPL), and the cells were cultured in the culture medium in a total volume of 2 ml.

The compounds of Examples were completely dissolved in DMSO and used in the experiment, and thymidine was completely dissolved in DW and used in the experiment. For thymidine block, the products were treated with 2 mM of thymidine (Sigma-Aldrich Cat. No. T9250-5G) and then incubated for 24 hours.

For release and chemical treatment, the medium was suctioned and washed 3 times with 1×PBS. Complete media was added, followed by incubation for 4 hours in a CO₂ incubator. Each compound was treated according to the concentration of 100 nM and then incubated for 6 hours again.

3. Western Blotting

For SDS-PAGE and Western blotting, 1×RIPA lysis buffer (Rockland, Cat. No. MB-030-0050; Lot no. 39751), 100× Protease Inhibitor Cocktail (Quartett, Cat. No. PPI1015; Lot no. PC050038424), Pierce

BCA protein assay kit (ThermoScientific, Cat. No. 23225; Lot no. UC276876), albumin standard (ThermoScientific, Cat. No. 23209; Lot no. UB269561), 4-15% Mini-PROTEAN TGX stain-free gel (Bio-rad, Cat. No. 4568085; Lot no. L007041B), 10× Tris/Glycine/SDS buffer (Bio-rad, Cat. No. 1610732; Lot no. 10000044375B); 10×TBS (Bio-rad, Cat. No. 1706435; Lot no. 1000045140B), 10% Tween 20 (Cat. No. 1610781; Lot no. L004152B), Color protein standard broad range (NEB, Cat. No. P7719S; Lot no. 10040349), 4× Laemmli sample buffer (Bio-rad, Cat. No. 1610747; Lot no. L004133B), β-mercaptoethanol (Sigma-Aldrich, Cat. No. M3148; Lot no. 60-24-2), SuperBlock

T20 (TBS) blocking buffer (ThermoScientific, Cat. No. 37536; Lot no. UC282578), 1M sodium azide solution (Sigma-Aldrich, Cat. No. 08591-1 mL-F; Lot no. BCBV4989), α-Rabbit pAb to Ms IgG (abcam, Cat. No. ab97046; Lot no. GR3252115-1), α-Goat pAb to Rb IgG (CST, Cat. No. 7074S; Lot no. 28), α-GAPDH (abeam, Cat. No. ab8245; Lot no. GR3275542-2), α-Plk1 (CST, Cat. No. 208G4), α-BRD4 (CST, Cat. No. 13440S), ECL

Prime western blotting reagents (GE Healthcare, Cat. No. RPN2232; Lot no. 17001655), Ponceau S solution (Sigma-Aldrich, Cat. No. P7170; Lot no. SLBV4112), Difco

Skim milk (BD, Cat. No. 232100; Lot no. 8346795), and iBlot® 2 NC Regular stacks (Invitrogen, Cat. No. IB23001; Lot no. 2NR110619-02) were used.

For cell harvesting, the cells were first separated from the plate using trypsin and then washed with the medium and PBS. Specifically, the medium was suctioned off and washed with 1 mL of PBS, and PBS was suctioned off. The cells were treated with 0.5 mL TrypLE

Express at 37° C. for 7 minutes to separate the cells, and then 0.5 mL

of complete medium was added to collect 1 mL of cell culture solution. Then, 1 mL of the cell collection solution was centrifuged at 8,000 rpm for 120 seconds, and the supernatant was removed. After washing with 0.2 mL of PBS, the PBS was removed.

For cell lysis, a lysis buffer was added and cell debris was removed to obtain a cell lysate. Specifically, the cells were treated with 70 μL of 1×RIPA buffer containing a protease inhibitor and incubated for 30 minutes on ice. Then, the cells were centrifuged at 4° C. and 15,000 rpm for 10 minutes to obtain a cell lysate.

Then, a standard curve was obtained using the BCA assay, and the protein mass in the lysate was quantified by substituting the curve equation. The mixture was incubated at 37° C. for 30 minutes using 20 μL of standard or sample solution, and 200 μL of BCA or Bradford solution, and measured at 562 nm absorbance. Samples were prepared by adding 4× sample buffer so that the quantity of protein added to each well was 15 μg.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by setting a running time of 100 minutes at 120 V on a 4-15% Mini-PROTEAN TGX stain-free gel (15 well). Transferring was performed on iBlot® 2 NC Mini stacks at PO mode of the dry blotting system. After staining using Ponceau S solution, blocking was performed for 1 hour with a blocking buffer (Thermo). After washing with 1×TBS containing 0.05% Tween 20, the product was reacted at 4° C. for 16 hours with anti-Plk1 (CST) antibody (1:500), anti-BRD4 (Cell signaling) antibody (1:1000) or anti-GAPDH (abcam) antibody (1:10,000) in 1×TBS-T as a primary antibody. After washing three times for 10 minutes with 1×TBS containing 0.05% Tween20, the product was reacted at room temperature for 1 hour with anti-mouse antibody (abcam) (1:10000) or anti-rabbit antibody (CST) (1:5000) in 1×TBS-T as a secondary antibody. Then, after washing three times for 10 minutes with 1×TBS containing 0.05% Tween 20, the product was detected with an ECL working solution (1:1).

To analyze the results, an image analyzer (GE) was used to obtain final blot data. The ratio of PLK1 to GAPDH for each sample was calculated using the ImageQuant TL (ver. 8.2.0) program. Each calculated value was entered into each cell of the Graphpad Prism 9 program, and the graph was automatically calculated to confirm the Dmax value corresponding to the protein degradation ability.

4. Confirmation of PLK1 Degradability of the Compounds of the Present Invention

As a result of the experiment, it was confirmed that all the compounds of the examples of the present invention exhibited a PLK1 degradability of 40 to 60%.

As a result of the above experiment, it was confirmed that the compounds of the present invention exhibited PLK1 protein degradability (Dmax) of 40% or more. In particular, the Dmax of compounds 2, 3, 7, 9 were 60% or more, indicating excellent PLK1 protein degradability. In addition, it was confirmed that the compounds of the present invention have remarkably excellent PLK1 protein degradability compared with Comparative Compounds 1 and 2 described in CN106543185 A, and compared with the Example 1 compound in which an aniline group was connected by a linker (FIG. 1 ).

The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. 

1. A compound represented by the following Formula I, or a seism stereoisomer thereof or a pharmaceutically acceptable salt thereof: ULM-Linker-PTM  [Formula I] in the Formula I above, ULM is CRBN or VHL E3 ubiquitin ligase binding moiety; PTM is PLK1 binding moiety represented by the following Formula II:

{in the Formula II above, R₁ is hydrogen, C₁₋₄ alkyl or —(C₁₋₃ alkyl)-OH; R₂ is —O—C₁₋₄ alkyl or —O—CF₃; R₃ is N or CH; and R₄ is NH, N(C₁₋₃ alkyl), CH₂ or CH(C₁₋₃ alkyl)}; and Linker is a chemical group that links ULM and PTM.
 2. The compound of claim 1, wherein ULM is CRBN E3 ubiquitin ligase binding moiety represented by the following Formula A:

wherein: X₁ is —CH₂—, —CH(C₁₋₄ alkyl)-, —CO— or —N═N—; and X₂ is hydrogen or C₁₋₃ alkyl.
 3. The compound of claim 1, wherein ULM is VHL E3 ubiquitin ligase binding moiety represented by the following Formula B:

wherein: {circle around (H)} is 5-membered heteroaryl; and Y₁ is hydrogen or C₁₋₃ alkyl.
 4. The compound of claim 1, wherein Formula II is represented by the following Formula III-1 or III-2.

wherein R₁, R₂, R₃ and R₄ are the same as defined in claim
 1. 5. The compound of claim 1, wherein the Linker is represented by the following Formula L:

wherein:

and ----- are each independently bond, L_(ULM) is covalently bonded to ULM moiety through

that is linked thereto, L_(PTM) is covalently bonded to PTM moiety through

that is linked thereto, L_(ULM) and L_(PTM) are each independently a single bond, —CH₂—, —NH—, —O—, —CO—, —OCO—, —CONH— or —NHCO—, L_(INT) is selected from —CH₂—, —CH₂CH₂—, —CHCH—, —CC—, —CH₂CH₂O—, —OCH₂CH₂—, —CH₂CH₂S—, —SCH₂CH₂—, —COO—, —CONH—, —NHCO— and {circle around (L)} {wherein {circle around (L)} is 3-10 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl, or 5-10 membered heteroaryl}.
 6. The compound of claim 1, wherein the compound is selected from compound 2 to
 12. 7. The compound of claim 1, wherein the compound is a bifunctional compound that induces PLK1 protein degradation.
 8. The compound of claim 1, wherein the compound induces selective PLK1 protein degradation.
 9. A composition for prevention or treatment of a PLK1 related disease comprising the compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
 10. The composition of claim 9, wherein the PLK1 related disease is one selected from a cancer, a benign tumor and a neurological disease. 